Researching COVID to enhance recovery (RECOVER) autopsy study protocol: Rationale, objectives, and design (preprint)

Importance: SARS-CoV-2 infection can result in ongoing, relapsing, or new symptoms or organ dysfunction after the acute phase of infection, termed Post-Acute Sequelae of SARS-CoV-2 (PASC), or long COVID. The characteristics, prevalence, trajectory and mechanisms of PASC are poorly understood. The objectives of the Researching COVID to Enhance Recovery (RECOVER) Tissue Pathology Study (RECOVER-Pathology) are to: (1) characterize prevalence and types of organ injury/disease and pathology occurring with PASC; (2) characterize the association of pathologic findings with clinical and other characteristics; (3) define the pathophysiology and mechanisms of PASC, and possible mediation via viral persistence; and (4) establish a post-mortem tissue biobank and post-mortem brain imaging biorepository. Methods: RECOVER-Pathology is a cross-sectional study of decedents dying at least 15 days following initial SARS-CoV-2 infection. Eligible decedents must meet WHO criteria for suspected, probable, or confirmed infection and must be aged 18 years or more at the time of death. Enrollment occurs at 7 sites in four U.S. states and Washington, DC. Comprehensive autopsies are conducted according to a standardized protocol within 24 hours of death; tissue samples are sent to the PASC Biorepository for later analyses. Data on clinical history are collected from the medical records and/or next of kin. The primary study outcomes include an array of pathologic features organized by organ system. Casual inference methods will be employed to investigate associations between risk factors and pathologic outcomes. Discussion: RECOVER-Pathology is the largest autopsy study addressing PASC among US adults. Results of this study are intended to elucidate mechanisms of organ injury and disease and enhance our understanding of the pathophysiology of PASC.

Decomposing the sources of SARS-CoV-2 fitness variation in the United States (preprint)

The fitness of a pathogen is composite phenotype determined by many different factors influencing growth rates both within and between hosts. Determining what factors shape fitness at the host population-level is especially challenging because both intrinsic factors like pathogen genetics and extrinsic factors such as host behaviour influence between-host transmission potential. These challenges have been highlighted by controversy surrounding the population-level fitness effects of mutations in the SARS-CoV-2 genome and their relative importance when compared against non-genetic factors shaping transmission dynamics. Building upon phylodynamic birth-death models, we develop a new framework to learn how hundreds of genetic and non-genetic factors have shaped the fitness of SARS-CoV-2. We estimate the fitness effects of all amino acid variants and several structural variants that have circulated in the United States between February and September 2020 from viral phylogenies. We also estimate how much fitness variation among pathogen lineages is attributable to genetic versus non-genetic factors such as spatial heterogeneity in transmission rates. Up to September 2020, most fitness variation between lineages can be explained by background spatial heterogeneity in transmission rates across geographic regions. Furthermore, no genetic variant including the Spike D614G mutation has had a significant effect on population-level fitness. Instead, the rapid increase in the frequency of the Spike D614G can be explained by the variant having a spatial transmission advantage due to first establishing in regions with higher transmission rates during the earliest stages of the pandemic.

Evaluation of in vitro activity of copper gluconate against SARS-CoV-2 using confocal microscopy-based high content screening (preprint)

ContextSevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that emerged late in 2019 is the etiologic agent of coronavirus disease 2019 (Covid-19). There is an urgent need to develop curative and preventive therapeutics to limit the current pandemic and to prevent the re-emergence of Covid-19. This study aimed to assess the in vitro activity of copper gluconate against SRAS-CoV-2. MethodsVero E6 cells were treated with copper gluconate 18 hours before infection. Cells were infected with a recombinant GFP expressing SARS-CoV-2. Infected cells were maintained in fresh medium containing copper gluconate for an additional 48-hour period. The infection level was measured by the confocal microscopy-based high content screening method. The cell viability in presence of copper gluconate was assessed by XTT assay. ResultsThe viability of Vero E6 cells treated with copper gluconate up to 200 M was found to be similar to that of untreated cells, but it dropped below 40% with 400 M of this agent. The infection rate was 23.8%, 18.9%, 20.6%, 6.9%, 5.3%,5.2% in cells treated with 0, 2, 10, 25, 50 and 100 M of copper gluconate respectively. As compared to untreated cells, the number of infected cells was reduced by 71%, 77%, and 78% with 25, 50, and 100 M of copper gluconate respectively (p < 0.05). ConclusionCopper gluconate was found to mitigate SARS-CoV-2 infection in Vero E6 cells. Furthers studies are needed to determine whether copper homeostasis could play a role in SARS-CoV-2 infection. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/422548v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1f99949org.highwire.dtl.DTLVardef@1bebae6org.highwire.dtl.DTLVardef@e05e37org.highwire.dtl.DTLVardef@49a3b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

A novel cell culture system modeling the SARS-CoV-2 life cycle (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19, and no effective antiviral agents and vaccines are available. SARS-CoV-2 is classified as a biosafety level-3 (BLS-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2-GFP/{Delta}N trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.

The Potential for SARS-CoV-2 to Evade Both Natural and Vaccine-induced Immunity (preprint)

SARS-CoV-2 attaches to the surface of susceptible cells through extensive interactions between the receptor binding domain (RBD) of its spike protein and angiotensin converting enzyme type 2 (ACE2) anchored in cell membranes. To investigate whether naturally occurring mutations in the spike protein are able to prevent antibody binding, yet while maintaining the ability to bind ACE2 and viral infectivity, mutations in the spike protein identified in cases of human infection were mapped to the crystallographically-determined interfaces between the spike protein and ACE2 (PDB entry 6M0J), antibody CC12.1 (PDB entry 6XC2), and antibody P2B-2F6 (PDB entry 7BWJ). Both antibody binding interfaces partially overlap with the ACE2 binding interface. Among 16 mutations that map to the RBD:CC12.1 interface, 11 are likely to disrupt CC12.1 binding but not ACE2 binding. Among 12 mutations that map to the RBD:P2B-2F6 interface, 8 are likely to disrupt P2B-2F6 binding but not ACE2 binding. As expected, none of the mutations observed to date appear likely to disrupt the RBD:ACE2 interface. We conclude that SARS-CoV-2 with mutated forms of the spike protein may retain the ability to bind ACE2 while evading recognition by antibodies that arise in response to the original wild-type form of the spike protein. It seems likely that immune evasion will be possible regardless of whether the spike protein was encountered in the form of infectious virus, or as the immunogen in a vaccine. Therefore, it also seems likely that reinfection with a variant strain of SARS-CoV-2 may occur among people who recover from Covid-19, and that vaccines with the ability to generate antibodies against multiple variant forms of the spike protein will be necessary to protect against variant forms of SARS-CoV-2 that are already circulating in the human population.

Generation of a SARS-CoV-2 Replicon as a Model System to Dissect Virus Replication and Antiviral Inhibition (preprint)

SARS-CoV-2 research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a non-infectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlights the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19.

SARS-CoV-2 RNA reverse-transcribed and integrated into the human genome (preprint)

Prolonged SARS-CoV-2 RNA shedding and recurrence of PCR-positive tests have been widely reported in patients after recovery, yet these patients most commonly are non-infectious1-14. Here we investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the human genome and that transcription of the integrated sequences might account for PCR-positive tests. In support of this hypothesis, we found chimeric transcripts consisting of viral fused to cellular sequences in published data sets of SARS-CoV-2 infected cultured cells and primary cells of patients, consistent with the transcription of viral sequences integrated into the genome. To experimentally corroborate the possibility of viral retro-integration, we describe evidence that SARS-CoV-2 RNAs can be reverse transcribed in human cells by reverse transcriptase (RT) from LINE-1 elements or by HIV-1 RT, and that these DNA sequences can be integrated into the cell genome and subsequently be transcribed. Human endogenous LINE-1 expression was induced upon SARS-CoV-2 infection or by cytokine exposure in cultured cells, suggesting a molecular mechanism for SARS-CoV-2 retro-integration in patients. This novel feature of SARS-CoV-2 infection may explain why patients can continue to produce viral RNA after recovery and suggests a new aspect of RNA virus replication.

KIM-1/TIM-1 is a Receptor for SARS-CoV-2 in Lung and Kidney (preprint)

SARS-CoV-2 precipitates respiratory distress by infection of airway epithelial cells and is often accompanied by acute kidney injury. We report that Kidney Injury Molecule-1/T cell immunoglobulin mucin domain 1 (KIM-1/TIM-1) is expressed in lung and kidney epithelial cells in COVID-19 patients and is a receptor for SARS-CoV-2. Human and mouse lung and kidney epithelial cells express KIM-1 and endocytose nanoparticles displaying the SARS-CoV-2 spike protein (virosomes). Uptake was inhibited both by anti-KIM-1 antibodies and by TW-37, our newly discovered inhibitor of KIM-1-mediated endocytosis. Enhanced KIM-1 expression by human kidney tubuloids increased uptake of virosomes. KIM-1 positive cells express less angiotensin-converting enzyme 2 (ACE2), the well-known receptor for SARS-CoV-2. Using microscale thermophoresis, the EC50 for KIM-1-SARS-CoV-2 spike protein, and receptor binding domain (RBD) interactions, were 19 and 10 nM respectively. Thus KIM-1 is an alternative receptor to ACE2 for SARS-CoV-2. KIM-1 targeted therapeutics may prevent and/or treat COVID-19.